Real-time detection of DNA amplification during the polymerase chain reaction (PCR) process provides quantitative data for amplifiable DNA target sequences by relating the number of temperature cycles during thermal cycling to reach a concentration threshold (Ct) of the target sequence to the amount of target DNA present at the beginning of the PCR process. The determination of the amount of target DNA present can be effected by detecting the accurate Ct.
High-throughput systems can provide DNA amplification of multiple samples in parallel, such as in a microwell tray or microcard. Assays can provide multiple DNA target sequences of interest, such as diagnostic assays for HIV screening. These assays can provide multiple spectrally distinguishable species, such as different fluorescent dyes, to detect Ct values for each of the multiple DNA target sequences of interest in each of the multiple samples thermally cycled in parallel.
Spectral non-uniformity between multiple samples thermally cycled in parallel at different locations on the tray and/or multiple dyes at the same location on the tray can cause repeatable systematic Ct error. Spectral non-uniformity can result from spectral variation in excitation light provided to the samples, spectral variations due to optical components, such as filters and beam splitters, and/or spectral variations in the detector. It can be desirable to reduce the effects of spectral non-uniformity between the multiple samples thermally cycled in parallel by calibrating the detection of light from the biological samples by determining a correction factor to more accurately detect Ct values.